The gram-positive bacillus Listeria monocytogenes predominantly infects immunocompromised patients, causing bacteremia and meningitis while the gram-negative bacillus Shigella flexneri infects normal hosts causing severe diarrhea and dehydration. The pathogenesis of Listeriosis and Shigellosis absolutely requires these intracellular bacteria to usurp the host cell's contractile system. Listeria and Shigella induce host cell actin to assemble into rocket tails that rapidly propel the bacteria through the cytoplasm, allowing their cell-to-cell spread and avoidance of the humoral immune system. Actin assembly occurs in a discrete polymerization zone directly behind the motile bacteria. This region blocks the host cell actin-regulatory proteins, gelsolin, CapZ and CapG, that normally cap the fast growing ends of actin filaments. This blocking activity allows actin filaments to rapidly assemble in this discrete zone. Two of these proteins, gelsolin and CapG, require micromolar calcium to function. We will: Aim I - Elucidate how Listeria blocks barbed end-capping proteins in the polymerization zone. Pyrenyl actin and right angle light scattering will be used to examine how profilin combined PIP2 and VASP or N-WASP effects actin filament capping by CapG, CapZ and gelsolin. Capping inhibition by Listeria will be investigated in brain cell free extracts before and after depletion of profilin and VASP. Localization of PIP2 (well known to block capping activity) in Listeria and Shigella infected cells will be studied using a GFP labeled probe. The effects of blocking PIP2 production using the PI kinase inhibitors Wortmannin and quercetin, infecting cells with Listeria ActA mutants lacking PIP2 binding sites, and ActA mutants lacking VASP binding sites will be examined. Aim II - Study the Calcium-Dependence of Listeria and Shigella actin-based motility. Calcium is a critical signal for turning on and off actin regulatory proteins, and we have found that the chelator BAPTAM blocks Shigella actin-based motility and slows the disassembly of Listeria rocket tails. The Ca2+-sensitivity of N-WASP and vinculin, cell proteins unique to Shigella-induced actin assembly, as well as gelsolin will be studied. These investigations should clarify key regulatory pathways required for Listeria- and Shigella-induced actin assembly and may identify new therapeutic targets for treating Listeriosis and Shigellosis.